During the last fiscal year, the NIH SCCF has made progress in a number of areas as highlighted below. In collaboration with NIH-CRM, 5 transgenic hESC lines, which express traceable markers from cell type-specific promoters, were generated and deposited with WiCell for distribution. This manuscript was published in 2015. To study the role of individual genes in human disease pathogenesis, two inducible model cell lines expressing ALPHA-SYNUCLEIN or mutant LAMIN-A (PROGERIN) were generated in H9 (WA09) hESCs using a tetracycline-inducible system. Overexpression of each transgene models Parkinson's disease or Hutchinson-Gilford Progeria syndrome respectively with the level of transgene expression, regulated by the inducible system, anticipated to mimic the severity of the disease symptoms. However, analysis of transgene expression showed that the level of expression induced by the drug was not consistent within the cell population, possibly due to random integration of the transgene. This work was presented at the International Society for Stem Cell Research (ISSCR) annual meeting in 2015. To overcome this problem, another set of tetracycline-inducible lines have been generated in WA09 and an induced pluripotent stem cell (iPSC) line by targeting safe-harbor sites for integration. Analysis is underway. We have continued to develop protocols to optimize human PSC culture on different extracellular matrices, particularly regarding the use of laminin-based methods to facilitate definitive endoderm differentiation and hepatic lineage maturation. This work was also presented at the ISSCR annual meeting in 2015. Other assays in development include the use of multiple panel surface markers in monitoring cellular differentiation and functional maturation and glucose metabolism assays to monitor cellular energetic states in hPSC culture and differentiation. In terms of bringing pluripotent stem cells to the clinic, we have been evaluating novel xeno-free substrates, media and small molecule inhibitors as well as non-integrating methods of reprogramming. We are advising on the establishment of GMP protocols related to Dr. Kapil Bhartis (NEI) clinical initiative regarding iPSC derivation and differentiation into retinal pigmented epithelial cells. Finally, we continued to mentor and teach standard and feeder-free, pluripotent stem cell culture, provided assistance and advise on the generation of iPSCs from collaborators samples, as well as assistance and advise on differentiation strategies as requested. Such differentiation strategies have included the derivation of human intestinal organoids for pilot testing culture of gut pathogens as well as cortical spheroids to model brain development. We have also assisted collaborators by providing material and neural stem cell lines derived from PSCs, resulting in the publication of 2 other manuscripts in 2015. As always, we update the SCU website with protocols and information as it becomes available to aid other researchers in their studies.